© 2008 The Author(s).
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RESEARCH PAPER |
Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue
1Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México DF 04510, México
2Division of Biochemistry, 105 Life Sciences Center, 1201 E. Rollins, Columbia, MO 65211, USA
3Departamento de Biología Comparada, Facultad de Ciencias, Universidad Nacional Autónoma de México, México DF 04510, México
To whom correspondence should be addressed. E-mail: fcg{at}servidor.unam.mx
After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana.
Key words: Kunitz proteinase inhibitor homologue, Nicotiana, pollen–pistil interactions, pollination
* These authors contributed equally to this work
Received 26 March 2008; Revised 29 May 2008 Accepted 3 June 2008